The heat shock response of Drosophila melanogaster is characterized by the rapid and robust activation of the major heat shock genes. Although much is known about heat shock promoter architecture and the transcription factors which recognize the promoters, the mechanistic detail of heat shock gene transcription remain unclear. Two transcription factors, GAGA factor (GAF) and TATA-binding protein, and RNA polymerase II (Pol II) occupy the uninduced heat shock promoters. Pol II, although capable of producing small transcripts, is paused at the heat shock promoter. It is thought that the protein-protein and protein-DNA interactions among these factors play an important role in the maintenance of the open chromatin structure at the promoter and in retention of the paused polymerase. To address this hypothesis, DNA-binding domain and interaction domain mutants of GAF will be constructed and subjected to rigorous in vivo and in vitro analysis. Given the similarities between heat shock promoter sequences and those of other promoters, and the presence of a paused polymerase on several non-heat shock genes in Drosophila and in mammalian cells, studies with these GAF mutants may provide a general understanding of promoter architecture, polymerase pausing, and gene activation.